Specific and sensitive determination of lipid hydroperoxides with chemical derivatization into 1-naphthyldiphenylphosphine oxide and high-performance liquid chromatography

A highly sensitive and specific high-performance liquid chromatographic (HPLC) method to determine the hydroperoxide level in biological samples involving the oxidation of 1-naphthyldiphenylphosphine into its oxide was developed. The detection limit of the method applied to the determination of cumene hydroperoxide was 1 pmol and the yield of the phosphine oxide based on the hydroperoxide in the concentration range from 0.2 μM to 1 mM was 85–100%. The linearity between the amount of the hydroperoxide and the HPLC peak height was excellent in a wide range (1 pmol to 5 nmol). The recoveries of cumene hydroperoxide from saline and homogenates of mouse brain, heart, kidney and liver were 88.5 ± 10, 83.3 ± 13, 75.3 ± 1.0, 46.3 ± 13.4 and 18.7 ± 10%, respectively. The recovery of exogenously added lecithin hydroperoxide from mouse liver homogenate was 52.4 ± 9.8%, while that from saline was 63.6 ± 5.4%. The peroxide levels in the brain, heart, kidney and liver of 5-week-old mice were determined to be 474 ± 170, 426 ± 183, 655 ± 121 and 239 ± 31 pmol/mg protein, respectively.